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PloS One 2024Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt...
Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients. However, some aspects of the assay, such as sensitivity and time cost, need improvement. Here, we developed an express test, which may be potentially used in clinical practice for the assessment of serum-caused SARS-CoV-2 inhibition in infected cell cultures. It implies the detection and counting of coronaviral fluorescent-forming units (FFU) and includes two sequentially used developing components: biotinylated mouse monoclonal antibodies against the recombinant N protein of SARS-CoV-2 (B.1) and the recombinant EGFP-streptavidin fusion protein. Due to the universal specificity of the antibodies, our analytical tool is suitable for the detection of various strains of SARS-CoV-2 when determining both the infectious titer of viruses and the titer of serum virus-neutralizing antibodies. The developed two-component test system is characterized by high sensitivity, a reduced number of analytic stages and low assay cost, as well as by flexibility, since it may be modified for detection of other pathogens using the appropriate antibodies.
Topics: SARS-CoV-2; Humans; COVID-19; Animals; Antibodies, Viral; Vero Cells; Chlorocebus aethiops; Mice; Fluorescent Antibody Technique; Antibodies, Monoclonal; Antibodies, Neutralizing
PubMed: 38820303
DOI: 10.1371/journal.pone.0304534 -
Frontiers in Immunology 2024Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in...
Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.
Topics: Humans; Female; Male; Child; Palatine Tonsil; Adult; Influenza Vaccines; Influenza, Human; Sex Characteristics; Child, Preschool; Adolescent; Antibodies, Viral; Memory B Cells; Organ Specificity; Young Adult; Sex Factors; CD4-Positive T-Lymphocytes; B-Lymphocytes; Immunologic Memory
PubMed: 38812520
DOI: 10.3389/fimmu.2024.1373537 -
Frontiers in Bioscience (Landmark... May 2024To investigate the immune responses and protection ability of ultraviolet light (UV)-inactivated recombinant vesicular stomatitis (rVSV)-based vectors that expressed a...
BACKGROUND
To investigate the immune responses and protection ability of ultraviolet light (UV)-inactivated recombinant vesicular stomatitis (rVSV)-based vectors that expressed a fusion protein consisting of four copies of the influenza matrix 2 protein ectodomain (tM2e) and the Dendritic Cell (DC)-targeting domain of the Ebola Glycoprotein (EΔM), (rVSV-EΔM-tM2e).
METHOD
In our previous study, we demonstrated the effectiveness of rVSV-EΔM-tM2e to induce robust immune responses against influenza M2e and protect against lethal challenges from H1N1 and H3N2 strains. Here, we used UV to inactivate rVSV-EΔM-tM2e and tested its immunogenicity and protection in BALB/c mice from a mouse-adapted H1N1 influenza challenge. Using Enzyme-Linked Immunosorbent Assay (ELISA) and Antibody-Dependent Cellular Cytotoxicity (ADCC), the influenza anti-M2e immune responses specific to human, avian and swine influenza strains induced were characterized. Likewise, the specificity of the anti-M2e immune responses induced in recognizing M2e antigen on the surface of the cell was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis.
RESULTS
Like the live attenuated rVSV-EΔM-tM2e, the UV-inactivated rVSV-EΔM-tM2e was highly immunogenic against different influenza M2e from strains of different hosts, including human, swine, and avian, and protected against influenza H1N1 challenge in mice. The FACS analysis demonstrated that the induced immune responses can recognize influenza M2 antigens from human, swine and avian influenza strains. Moreover, the rVSV-EΔM-tM2e also induced ADCC activity against influenza M2e from different host strains.
CONCLUSIONS
These findings suggest that UV-inactivated rVSV-EΔM-tM2e could be used as an inactivated vaccine against influenza viruses.
Topics: Animals; Influenza Vaccines; Influenza A Virus, H1N1 Subtype; Ultraviolet Rays; Mice, Inbred BALB C; Orthomyxoviridae Infections; Female; Mice; Humans; Viral Matrix Proteins; Vesiculovirus; Vaccines, Inactivated
PubMed: 38812326
DOI: 10.31083/j.fbl2905195 -
PloS One 2024Rapid syphilis testing plays a crucial role in global health strategies, addressing the urgent need for prompt and accurate diagnostics, especially in settings with...
Rapid syphilis testing plays a crucial role in global health strategies, addressing the urgent need for prompt and accurate diagnostics, especially in settings with limited resources. Despite their practical utility, these tests often lack thorough validation, leading to concerns about their efficacy and reliability. This study aims to evaluate two prototypes of the Onsite Syphilis Ab Combo Rapid Test (Fd and Ff) and compare their performance with the established chemiluminescent microparticle immunoassay (CMIA) method. Employing a reverse algorithm approach, the study analyzed 450 serum samples, including those from syphilis patients, healthy individuals, and cases with potential cross-reactions. Results of the rapid test kit were then correlated with CMIA findings, RPR, and TPPA titers. The results showed that prototype Fd exhibited a sensitivity of 100.0%, specificity of 98.8%, positive predictive value (PPV) of 8.4%, negative predictive value (NPV) of 100.00% and accuracy of 98.8%. Similarly, prototype Ff exhibited sensitivity of 100.0%, but with a slightly higher specificity of 99.6%, PPV of 21.5%, NPV of 100.0% and accuracy of 99.6%. Moreover, both prototypes Fd and Ff of the Onsite Syphilis Ab Combo Rapid Test demonstrated significant efficacy diagnostic tool, offering clear and straightforward interpretation for clinicians in varied CMIA, RPR and TPPA titer scenarios. The Onsite Syphilis Ab Combo Rapid Test prototypes, Fd and Ff, demonstrated high sensitivity and specificity, comparable to CMIA methods. The effectiveness highlights their suitability for syphilis screening, particularly in non-laboratory settings or situations requiring immediate results. The validation of these prototypes supports their integration into current syphilis diagnostic algorithms, potentially contributing to improved public health outcomes.
Topics: Humans; Treponema pallidum; Syphilis; Reagent Kits, Diagnostic; Antibodies, Bacterial; Syphilis Serodiagnosis; Sensitivity and Specificity; Male; Female; Adult; Middle Aged; Immunoassay; Reproducibility of Results; Rapid Diagnostic Tests
PubMed: 38809884
DOI: 10.1371/journal.pone.0303477 -
Applied Microbiology and Biotechnology May 2024The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to...
The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.
Topics: Animals; Antibodies, Monoclonal; African Swine Fever Virus; Epitope Mapping; Enzyme-Linked Immunosorbent Assay; Antibodies, Viral; Swine; African Swine Fever; Mice; Capsid Proteins; Mice, Inbred BALB C; Sensitivity and Specificity; Epitopes
PubMed: 38809284
DOI: 10.1007/s00253-024-13146-x -
Revista Da Sociedade Brasileira de... 2024Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a...
BACKGROUND
Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors.
METHODS
We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%).
CONCLUSIONS
Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.
Topics: Humans; Sensitivity and Specificity; Antibodies, Fungal; Immunodiffusion; Paracoccidioidomycosis; Paracoccidioides; Reagent Kits, Diagnostic; Female; Male
PubMed: 38808801
DOI: 10.1590/0037-8682-0094-2024 -
Frontiers in Cellular and Infection... 2024Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic...
INTRODUCTION
Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity.
METHODS
The study involved 300 subjects, whose infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA.
RESULTS
Of the 300 subjects, 171 (57%) had infection, with 165 of the 171 (96.5%) being positive for either or both of the allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons.
DISCUSSION
These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the gene.
Topics: Plasmodium falciparum; Antigens, Protozoan; Ghana; Humans; Protozoan Proteins; Malaria, Falciparum; Membrane Proteins; Antibodies, Protozoan; Female; Adult; Male; Adolescent; Young Adult; Child; Genetic Variation; Child, Preschool; Middle Aged; Sequence Analysis, DNA; Enzyme-Linked Immunosorbent Assay; Polymerase Chain Reaction; Antigenic Variation; DNA, Protozoan
PubMed: 38808064
DOI: 10.3389/fcimb.2024.1375249 -
Frontiers in Immunology 2024COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our...
INTRODUCTION
COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs).
METHODS
Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay.
RESULTS
The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2 COVID-19 vaccination, but were restored upon the 3 vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination.
DISCUSSION
Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.
Topics: Humans; Antibodies, Viral; COVID-19; Memory B Cells; SARS-CoV-2; COVID-19 Vaccines; Immunity, Humoral; Male; Adult; Cross Reactions; Female; Plasma Cells; Middle Aged; Immunoglobulin G; Vaccination; Influenza Vaccines; Immunologic Memory; Antibodies, Neutralizing; Epitopes, B-Lymphocyte; B-Lymphocytes; Spike Glycoprotein, Coronavirus; Kinetics
PubMed: 38807606
DOI: 10.3389/fimmu.2024.1382911 -
Communications Biology May 2024Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate...
Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate sensitivity and coverage of current procedures. Herein, we introduce a super-sensitivity and specificity technique for detecting ctDNA mutations, named HiCASE. The method utilizes PCR-based CRISPR, coupled with the restriction enzyme. In this work, HiCASE focuses on testing a series of EGFR mutations to provide enhanced detection technology for non-small cell lung cancer (NSCLC), enabling a detection sensitivity of 0.01% with 40 ng cell free DNA standard. When applied to a panel of 140 plasma samples from 120 NSCLC patients, HiCASE exhibits 88.1% clinical sensitivity and 100% specificity with 40 μL of plasma, higher than ddPCR and Super-ARMS assay. In addition, HiCASE can also clearly distinguish T790M/C797S mutations in different positions at a 1% variant allele frequency, offering valuable guidance for drug utilization. Indeed, the established HiCASE assay shows potential for clinical applications.
Topics: Humans; Circulating Tumor DNA; Mutation; ErbB Receptors; Lung Neoplasms; Carcinoma, Non-Small-Cell Lung; CRISPR-Cas Systems; Sensitivity and Specificity; DNA Mutational Analysis; Female; Male
PubMed: 38806596
DOI: 10.1038/s42003-024-06368-2 -
Antibodies (Basel, Switzerland) May 2024The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical...
The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.
PubMed: 38804307
DOI: 10.3390/antib13020039